This invention relates to a gene encoding cellulose synthase complex originating in Acetobacter xylinum subsp. sucrofermentans, a gene encoding cellulase, a gene encoding xcex2-glucosidase (G3ase), and gene group comprising these genes, as well as a novel xcex2-glucosidase (G3ase) itself.
It is well known that UDP-glucose is a direct substrate in cellulose biosynthesis of Acetobacter, which is linked together by a membrane protein complex called xe2x80x9ccellulose synthasexe2x80x9d, and released out of cells. This complex has been reported to consist of four proteins encoded by an operon of cellulose synthase gene, being named bcs A, B, C and D, respectively (H. C. Wong, et al., P.N.A.S., Vol.87, pgs.8130-8134 (1990)). Among these genes, bcsA, B and C genes are known to be essential for cellulose synthesis since their destruction would lose cellulose-producing capacity. It has been reported that bcsD gene also plays an important role since its destruction would cause a significant change of the structure of cellulose (I. M. Saxena, et al., J.Bacteriol., Vol.176, pgs.5735-5752 (1994)). Recently, it has been reported that the second cellulose synthase gene operon was obtained (I. M. Saxena, et al., J.Bacteriol., Vol.177, pgs.5276-5283 (1995)).
The cellulose synthase complex needs di-GMP as a cofactor. di-GMP is synthesized by cyclase and a gene encoding this enzyme has also been reported (R. Tal and D. H. Gelfand, PCT WO93/11244 (1994)).
It has been reported that upstream of the cellulose synthase gene operon, there are a cellulase gene (CMCase) and another gene (R. Standal, et al., J.Bacteriol., Vol.176, pgs.665-672 (1994)).
The present inventors have studied the cellulose synthase complex gene operon of Actobacter, and now succeeded in determinating the base sequences of a series of genes comprising a novel cellulose synthase complex gene operon and cellulase gene, which originate in Acetobacter xylinum subsp. sucrofermentans. According to our examination of the base sequence of a novel gene downstream of the novel cellulose synthase complex gene operon, we have also found that the nobel gene conserves well the sequence/region that are maintained in xcex2-glucosidase of various organisms (Y. Kashiwagi, et al., J.Ferment.Bioeng., Vol.78, pgs.394-398 (1994)) and therefore confirmed that this gene is xcex2-glucosidase.
Further, we have actually purified a protein encoded by the above xcex2-glucosidase gene, and examined its various properties.
The present invention relates to a gene encoding a protein constituting a cellulose synthase complex originating in Acetobacter xylinum subsp. sucrofermentans, particularly to a gene encoding a protein having an amino acid sequence represented by one of SEQ ID NO:2xcx9cSEQ ID NO:5.
This invention also relates to a gene encoding a variant protein having cellulose synthase activity and having an amino acid sequence that has been partially changed from that represented by one of SEQ ID NO:2xcx9cSEQ ID NO:5 by deletion, replacement or addition of one or a few amino acids. The genes of the present invention are therefore not limited to those originating in Acetobacter xylinum subsp. sucrofermentans. 
There may be exemplified as the base sequences of said genes"" DNA those shown as bcsA, bcsB, bcsC, and bcsD in SEQ ID NO:1. Furthermore, the present invention includes any base sequence or any part thereof prepared by a chemical synthesis and genetic engineering method by taking degeneracy of a genetic codon into consideration, which base sequence encodes the same amino acid sequence.
Also the present invention includes a gene comprising DNA that may hybridize with the above base sequences under stringent conditions, and encode the protein having cellulose synthase activity.
Further, the present invention relates to to a gene encoding cellulase originating in Acetobacter xylinum subsp. sucrofermentans, particularly to a gene encoding a protein having an amino acid sequence represented by SEQ ID NO:6.
This invention also relates to a gene encoding a variant protein having cellulase activity and having an amino acid sequence that has been partially changed from that represented by SEQ ID NO:6 by deletion, replacement or addition of one or a few amino acids. The gene of the present invention is not limited to that originating in Acetobacter xylinum subsp. sucrofermentans. 
There may be exemplified as the base sequence of said gene""s DNA that shown as CMCase in SEQ ID NO:1. Furthermore, the present invention includes any base sequence or any part thereof prepared by a chemical synthesis and genetic engineering method by taking degeneracy of a genetic codon into consideration, which base sequence encodes the same amino acid sequence.
Also the present invention includes a gene comprising DNA that may hybridize with the above base sequences under stringent conditions, and encode the protein having cellulase activity.
Further, the present invention relates to xcex2-glucosidase (G3ase) originating in Acetobacter microorganisms such as Acetobacter xylinwn subsp. sucrofermentans, particularly to a protein having an amino acid sequence represented by SEQ ID NO:7.
The amino acid sequence of this protein is not limited to that of SEQ ID NO:7, but may include that of a variant protein having xcex2-glucosidase activity and having an amino acid sequence that has been partially changed from that represented by SEQ ID NO:7 by deletion, replacement or addition of one or a few amino acids.
This invention also relates to a gene encoding xcex2-glucosidase. One of its example is the base sequence shown as xcex2-glucosidase in SEQ ID NO:1. Furthermore, the present invention includes any base sequence or any part thereof prepared by a chemical synthesis and genetic engineering method by taking degeneracy of a genetic codon into consideration, which encodes the same amino acid sequence.
Also the present invention includes a gene comprising DNA that may hybridize with the above base sequence under stringent conditions, and encode the protein having xcex2-glucosidase activity.
One of the representatives of Acetobacter xylinum subsp. sucrofermentans of the present invention is BPR 2001, which has been deposited at the National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305 Japan) on Feb. 24, 1993 under the accession number FERM P-13466, and then transferred on Feb. 7, 1994 to the deposit under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and Regulation under the accession number FERM BP-4545.
Other examples of the microorganisms belonging to Acetobacter may be Acetobacter xylinum ATCC23768, Acetobacter xylinum ATCC23769, Acetobacter pasteurianus ATCC10245, Acetobacter xylinum ATCC14851, Acetobacter xylinum ATCC11142, Acetobacter xylinum ATCC10821; and the like.
The present invention further relates to a gene group comprising the gene (operon) encoding the cellulose synthase complex of the present invention, and the gene encoding xcex2-glucosidase originating in Acetobacter microorganisms downstream (on 3xe2x80x2-terminal side) of the cellulose synthase gene.
The present gene group may include the cellulase gene of the present invention and/or glucanase gene upstream of the cellulose synthase complex gene (operon). The present gene group may include further various structural genes and regulating genes such as a promoter and operator. Each of these genes is separated by an appropriate number of bases apart from the other genes. For example, xcex2-glucosidase gene of BPR 2001 strain is located 214-bp downstream of the gene encoding bcsD of the cellulose synthase complex. One embodiment of the base sequence of the present gene group is shown as SEQ ID NO:1. The genes comprised in said gene group, their locations in the base sequence, and intervals therebetween are shown in FIG. 1.
There exists an open reading frame (ORF2) of another gene between the cellulase gene and the bcsA gene of the cellulose synthase complex in the gene group of SEQ ID NO:1. The amino acid sequence encoded by the ORF2 is shown in SEQ ID NO:8. The function of a protein having this amino acid sequence has not been identified yet. However, it has been reported that the destruction of a gene which is located in the similar position in another bacterium would deteriorate its biosynthesis of cellulose. Accordingly, it is very likely assumed that the gene shown by the ORF2 is involved in the biosynthesis of cellulose.
It is considered that the genes and gene group of the present invention encode a series of enzymes that is essential for the production of the cellulose in Acetobacter, and that the gene group of the present invention may possibly be a transcription unit being regulated by a series of promoters.
The genes and gene group of the present invention may be prepared by methods known to those skilled in the art.
For example, a gene library is prepared from the DNAs of a strain of Acetobacter xylinum subsp. sucrofermentans by a known method. On the other hand, a primer is synthesized based on the base sequence of the gene encoding a known cellulose synthase. PCR method is then carried out using the above gene library as a template to give the genes of the present invention. The genes of the present invention may be alternatively prepared by a hybridization method using an amplified DNA fragment obtained by the PCR method or a probe DNA prepared based on the base sequence of the above DNA fragment.
Those skilled in the art may easily prepare the genes of the present invention by a chemical synthesis based on the base sequence or amino acid sequence encoded thereby of the genes disclosed in the present specification.
Accordngly, each gene constituting the gene group of the present invention does not necessarily originate in the same microorganism (strain). The gene group of the present invention may therefore be prepared by optionally linking genes by a genetic engineering method, each of which genes has different origins and separately prepared.
Those genes and gene group may be inserted into a host cell such as E.coli to produce a series of the genes required for the production of cellulose.
The present invention is therefore related also to an expression vector comprising the gene(s) or the gene group, to a transformed cell such as E.coli transformed with the expression vector.
The expression vector of the present invention may optionally comprise an enhancer, promoter, ribosome-binding sequence, signal peptide-encoding sequence, replication origin and gene encoding a selection marker in addition to the above genes and gene group.
The expression vector, the transformed cell, and a series of the enzymes essential for the production of celulose may be prepared by various genetic engineering methods known to those skilled in the art.
Accordingly, the present invention relates further to the thus prepared recombinant proteins, i.e., the above enzymes.